This proposal focuses on the mechanism of action of the hormone erythropoietin (EPO) in erythroid cells. The aim of the laboratory has shifted from the structure of the EPO receptor (EPOR) to the posttranslational modification of the EPOR, signaling of proliferation and viability, and the mechanisms that active apoptosis. The hypothesis that an erythroid cell can become more or less responsive to EPO due to phosphorylation of serine residues of the EPOR will be tested. This possibility is important in determining if the EPOR and related family of receptors can be modulated through intracellular signaling to become hyper-responsive to hormones and growth factors in cancers and hyper-proferative diseases. EPOR mediates its actions by interacting with the tyrosine protein kinase, JAK2. Preliminary evidence shows that the EPOR phosphorylated on serine residues selectively binds to the JAK2 kinase in vivo and in vitro. Cells that are 100-fold more responsive to EPO have a large fraction of JAK2 preassociated with the EPOR. The first aim of this proposal will be to test the interaction of JAK2 in cells with varied responsiveness to EPO in vivo and in vitro. Biochemical experiments will determine which sites are phosphorylated. EPOR molecules in which the serines are phosphorylated will deleted will be tested for ability to interact with JAK2 and will be tested for function. The second aim will be to test our hypothesis that there are two distinct signaling pathways in erythroid cells that separately control proliferation and viability (protection from apoptosis). The signals mediated by EPO that protect cells from apoptosis by maintaining Bcl-XI expression and phosphorylation of BAD will be studied. PI3- kinase will be tested for roles in proliferation and survival. Studies using dominant negative and estrogen activated forms of MAP kinase will test the hypothesis that MAP kinase is important in proliferation. The role of autocrine TNF alpha secretion in erythroid cells on proliferation and activation of JNK will be studied. The role of JNK in proliferation will be tested by dominant negative forms.